Biology subjects

Wills, J. W.

Publications and source records attributed to Wills, J. W..

8 recordsLinked to original sources

bioRxiv2026

Mutagenic and carcinogenic potency determinations for NDMA support the cumulative dose assumption underpinning the less-than-lifetime Threshold of Toxicological Concern

The management of N-nitrosamine impurities challenges pharmaceutical development and regulation worldwide. Because most medicinal exposures are shorter than lifetime and absolute impurity exclusion is impossible, reliable approaches to define duration-specific intake limits are essential. On the premise that carcinogenic risk is proportional to cumulative dose, the Less-Than-Lifetime (LTL) Threshold of Toxicological Concern (TTC) framework defines progressively lower intake limits for mutagenic impurities over longer exposures. However, N-nitrosamines are currently treated as a cohort of concern, necessitating compound-specific evaluation placing reliance on in vivo mutagenicity assays for impurity qualifications. To better understand durational potency relationships and the application domain of the LTL-TTC, we apply benchmark dose (BMD) modelling to cumulative-dose-scaled transgenic rodent (TGR), error-corrected sequencing and rodent carcinogenicity datasets for N-nitrosodimethylamine (NDMA) obtained from the published literature. For TGR, cumulative-dose scaling better resolved liver as the most sensitive organ and reduced interstudy variability: liver BMDs spanned [~]80-fold in daily-dose units but only [~]20-fold when scaled to cumulative dose. Among closely-matched mouse liver gavage studies, cumulative-dose BMDs only varied by [~]2.5-fold across 1 to 28-day treatment regimens. Error-corrected sequencing also demonstrated parity, with acute-dose regimens producing mutation burdens near-identical (< 1.2-fold) to those cumulated from 28-day repeat-dose regimens. Comparable results were obtained from carcinogenicity datasets confirming proportionality-of-effect to cumulative dose. These findings empirically support the validity of the LTL-TTC concept. More broadly, they demonstrate that short-term in vivo mutagenicity assays can serve as reliable surrogates for lifetime carcinogenicity studies, strengthening the scientific and regulatory basis for duration-adjusted acceptable intakes for N-nitrosamine impurities.

pharmacology and toxicology↗

bioRxiv2025

Alignment between Duplex Sequencing and transgenic rodent mutation assay data in the assessment of in vivo NDMA-induced mutagenesis

The nitrosamine N-nitrosodimethylamine (NDMA) is a mutagen and rodent carcinogen that has been identified as a process impurity in some commercially available medicines, leading to market withdrawals and new impurity control measures. Error-corrected DNA sequencing techniques, such as Duplex Sequencing (DS), have error rates low enough to revolutionise genetic toxicology testing by directly measuring in vivo mutagenesis within days of exposure. Here, DS was performed on liver samples from an OECD-compliant, Transgenic Rodent Gene Mutation Assay (TGR) conducted under GLP standards. MutaMouse specimens were orally dosed with NDMA using either a repeat-dose 28-day regimen (0.02-4 mg/kg(bw)/day) or single bolus doses of either 5 or 10 mg/kg(bw) administered on day one. Dose-dependent increases in mutation frequency were detected by DS in liver, enabling a No-Observed Genotoxic Effect Level (NOGEL) of 0.07 mg/kg(bw)/day to be determined, supported by mechanistic analyses of trinucleotide mutation spectra. Benchmark dose (BMD) modelling determined similar BMD50 values from either DS or TGR, demonstrating concordance across the two techniques albeit with greater precision from DS due to smaller inter-animal variation. DS offers a fundamental change in mutagenicity assessments enabling more precise point-of-departure determinations with mechanistic clarity and 3Rs advantages compared to the standard TGR approach.

pharmacology and toxicology↗

bioRxiv2024

Cell Targeting and Adjuvant Activity of Dietary Titanium Dioxide

Food-grade titanium dioxide (fgTiO2) is a bio-persistent particle under intense regulatory scrutiny. Paradoxically, meaningful in vivo cellular accumulation has never been demonstrated: the only known cell reservoirs for fgTiO2 are graveyard intestinal pigment cells which are metabolically and immunologically quiescent. Here we identify major new immunocompetent cell targets of fgTiO2 in humans, most notably in the subepithelial dome region of intestinal Peyers patches. Using multimodal microscopy techniques with single-particle detection and per-cell / vesicle image analysis we achieved correlative dosimetry, quantitatively recapitulating human cellular exposures in the terminal ileum of mice fed a fgTiO2-containing diet. Epithelial microfold cells selectively funneled fgTiO2 into LysoMac and LysoDC cells with ensuing accumulation. Notwithstanding, proximity extension analyses for 92 protein targets revealed no measureable perturbation of cell signalling pathways. When chased with oral {Delta}aroA-Salmonella, pro-inflammatory signalling was confirmed, but no augmentation by fgTiO2 was revealed despite marked same-cell loading. Interestingly, Salmonella caused the fgTiO2-recipient cells to migrate basolaterally in the patch and, sporadically, to the lamina propria, thereby fully recreating the intestinal tissue distribution of fgTiO2 in humans. Immunocompetent cells that accumulate fgTiO2 in vivo are now identified and we demonstrate a mouse model that finally enables human-relevant risk assessments of ingested, bio-persistent (nano)particles.

pharmacology and toxicology↗

bioRxiv2023

An ovine model for investigation of the microenvironment of the male mammary gland

The specific biology of the male breast remains relatively unexplored in spite of the increasing global prevalence of male breast cancer. Delineation of the microenvironment of the male breast is restricted by the low availability of human samples and a lack of characterisation of appropriate animal models. Unlike the mouse, the male ovine gland persists postnatally. We suggest that the male ovine mammary gland constitutes a promising adjunctive model for the male human breast. In this study we evaluate the male ovine mammary gland microenvironment, comparing intact and neutered males. Assessment of the glandular histo-anatomy highlights the resemblance of the male gland to that of the neonatal female sheep and confirms the presence of rudimentary terminal duct lobular units. Irrespective of neutered status, cell proliferation in epithelial and stromal compartments is low in males, and cell proliferation in epithelial cells and in the intralobular stroma is significantly lower than in pubertal female sheep. Between 42% and 72% of the luminal mammary epithelial cells in the male gland express the androgen receptor, and expression is significantly reduced by neutering. Luminal epithelial cells within the intact and neutered male gland also express oestrogen receptor alpha, but minimal progesterone receptor expression is observed. The distrubution of mammary leukocytes within the ducts and stroma is similar in the female mammary gland of sheep and other species. Both macrophages and T lymphocytes are intercalated in the epithelial bilayer and are more abundant in the intralobular stroma than the interlobular stroma, suggesting that they may have a protective immunological function within the vestigial glandular tissue of the male sheep. Mast cells are also observed within the stroma. These cells cluster nearer the glandular tissue and are frequently located adjacent to blood vessels. The abundance of mast cells is significantly higher in intact males compared to neutered males, suggesting that hormone signalling may impact mast cell recruitment. In this study, we demonstrate the utility of the male ovine mammary gland as a model for furthering our knowledge of postnatal male mammary biology.

pathology↗

bioRxiv2023

In the murine and bovine maternal mammary gland signal transducer and activator of transcription 3 is activated in clusters of epithelial cells around the day of birth

Signal Transducers and Activators of Transcription (STATs) regulate mammary gland development. Here we investigate the expression of pSTAT3 in the murine and bovine mammary gland around the day of birth. We identify polarisation of mammary alveoli towards either a low- or high-proportion of pSTAT3 positive alveolar epithelial cells. We present localised colocation analysis applicable to other mammary studies where identification, quantification and interrogation of significant, spatially congregated events is required. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. This finding represents a new facet of mammary STAT3 biology meriting further functional investigation.

cell biology↗

bioRxiv2023

OPTIMAL: An OPTimsed Imaging Mass cytometry AnaLysis framework for Segmentation and Data Exploration

Analysis of Imaging Mass Cytometry (IMC) data and other low-resolution multiplexed tissue imaging technologies is often confounded by poor single cell segmentation and sub-optimal approaches for data visualisation and exploration. This can lead to inaccurate identification of cell phenotypes, states or spatial relationships compared to reference data from single cell suspension technologies. To this end we have developed the "OPTIMAL" framework to benchmark any approaches for cell segmentation, parameter transformation, batch effect correction, data visualisation/clustering and spatial neighbourhood analysis. Using a panel of 27 metal-tagged antibodies recognising well characterised phenotypic and functional markers to stain the same FFPE human tonsil sample Tissue Microarray (TMA) over 12 temporally distinct batches we tested several cell segmentation models, a range of different arcsinh cofactor parameter transformation values, five different dimensionality reduction algorithms and two clustering methods. Finally we assessed the optimal approach for performing neighbourhood analysis. We found that single cell segmentation was improved by the use of an Ilastik-derived probability map but that issues with poor segmentation were only really evident after clustering and cell type/state identification and not always evident when using "classical" bi-variate data display techniques. The optimal arcsinh cofactor for parameter transformation was 1 as it maximised the statistical separation between negative and positive signal distributions and a simple Z-score normalisation step after arcsinh transformation eliminated batch effects. Of the five different dimensionality reduction approaches tested, PacMap gave the best data structure with FLOWSOM clustering out-performing Phenograph in terms of cell type identification. We also found that neighbourhood analysis was influenced by the method used for finding neighbouring cells with a "disc" pixel expansion outperforming a "bounding box" approach combined with the need for filtering objects based on size and image-edge location. Importantly OPTIMAL can be used to assess and integrate with any existing approach to IMC data analysis and, as it creates .FCS files from the segmentation output, allows for single cell exploration to be conducted using a wide variety of accessible software and algorithms familiar to conventional flow cytometrists.

bioinformatics↗

bioRxiv2021

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25 - 5.0 {micro}g/mL) and/or carbendazim (0.8 - 1.6 {micro}g/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for mononucleates, binucleates, mononucleates with MN and binucleates with MN, respectively. Successful classifications of trinucleates (90%) and tetranucleates (88%) in addition to other or unscorable phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful ([≤] 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent dose regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

pharmacology and toxicology↗

bioRxiv2020

Developing mammary terminal duct lobular units have a dynamic mucosal and stromal immune microenvironment

The human breast and ovine mammary gland undergo a striking degree of postnatal development, leading to formation of terminal duct lobular units (TDLUs). In this study we interrogated aspects of sheep TDLU growth to increase understanding of ovine mammogenesis and as a model for the study of breast development. Mammary epithelial proliferation is significantly higher in lambs less than two months old than in peri-pubertal animals. Ki67 expression is polarized to the leading edge of the developing TDLUs. Intraepithelial ductal macrophages exhibit striking periodicity and significantly increased density in lambs approaching puberty. Stromal macrophages are more abundant centrally than peripherally. The developing ovine mammary gland is infiltrated by intraepithelial and stromal T lymphocytes that are significantly more numerous in older lambs. In the stroma, hotspots of Ki67 expression colocalize with large aggregates of lymphocytes and macrophages. Multifocally these aggregates exhibit distinct organization consistent with tertiary lymphoid structures. The lamb mammary gland thus exhibits a dynamic mucosal and stromal immune microenvironment and, as such, constitutes a valuable model system that provides new insights into postnatal breast development. Summary statementDevelopment of terminal duct lobular units in the sheep mammary gland involves distinct growth phases and macrophage and lymphocyte fluxes. Tertiary lymphoid structures are present subjacent to the mucosal epithelium.

developmental biology↗